WO2007105442A1 - Agent therapeutique pour un trouble de prise alimentaire ou un trouble de consommation d'eau - Google Patents

Agent therapeutique pour un trouble de prise alimentaire ou un trouble de consommation d'eau Download PDF

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Publication number
WO2007105442A1
WO2007105442A1 PCT/JP2007/053267 JP2007053267W WO2007105442A1 WO 2007105442 A1 WO2007105442 A1 WO 2007105442A1 JP 2007053267 W JP2007053267 W JP 2007053267W WO 2007105442 A1 WO2007105442 A1 WO 2007105442A1
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Prior art keywords
peptide
salt
amino acid
activity
test substance
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PCT/JP2007/053267
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English (en)
Japanese (ja)
Inventor
Masamitsu Nakazato
Motoo Yamasaki
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Kyowa Hakko Kogyo Co., Ltd.
University Of Miyazaki
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Priority to JP2008505024A priority Critical patent/JPWO2007105442A1/ja
Publication of WO2007105442A1 publication Critical patent/WO2007105442A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/303—Eating disorders, e.g. anorexia, bulimia

Definitions

  • the present invention relates to a therapeutic agent for eating disorders or drinking disorders containing, as an active ingredient, a peptide having an activity that promotes eating or drinking behavior, and activity of the peptide using the peptide
  • the present invention relates to a method for screening for a substance that inhibits or promotes or an agonist or antagonist for the receptor of the peptide.
  • the VGF gene was identified as a gene that increases upon nerve growth factor stimulation of rat PC12 cells (see Non-Patent Document 1), and then the human VGF gene was isolated (see Non-Patent Document 2).
  • the VGF gene-disrupted mice food intake was unchanged, weight loss, body fat loss, increased oxygen consumption, increased exercise capacity, and abnormal reproductive function. In particular, increased energy metabolism is recognized (see Non-Patent Document 3).
  • the VGF gene is expressed in the central, peripheral nervous system, endocrine and neuroendocrine cells, and its expression and distribution are neuropeptide Y, peptide ⁇ , and ghrelin that control feeding behavior and gastrointestinal motility. It is similar to the expression pattern of cholecystokinin and is present in the part related to energy metabolism (see Non-Patent Document 4).
  • VGF The protein encoded by the VGF gene (hereinafter referred to as VGF) is 615 amino acids in human and 617 amino acids in rat / mouse.
  • the amino acids 1 to 22 of VGF are signal peptides, and there are sequences that undergo processing such as cleavage and amidation by prohormone complexase.
  • VGF processing has been investigated mainly in rats, and rat VGF (598 _617) (numbers in parentheses indicate the position of the peptide sequence in the amino acid sequence of VGF.
  • rat VGF-derived peptides have been confirmed to exist (See Patent Document 5).
  • Rat VGF (556—617) Rat VGF (577-617), rat VGF (588-617), rat VGF (599-617), which are partial peptides, are administered directly to the hypothalamic paraventricular nucleus (PVN) of male rats.
  • Human VGF For peptides derived from human VGF, human VGF (23-62), human VGF (23_59), human VGF (26_62) (see Non-Patent Document 7), and human VGF (2 3-58) in human cerebrospinal fluid.
  • Patent Document 1 Pamphlet of International Publication No. 01/07477
  • Patent Document 2 Pamphlet of International Publication No. 02/82075
  • Non-Patent Document 1 Science, (USA), 1985, No. 229, No. 4711, p.39 3-395
  • Non-Patent Document 2 Genomics, (USA), 1997, No. 45, No. 2, p.443 -446
  • Non-Patent Document 3 Neuron, (USA), 1999, 23rd, No. 3, p.537—48
  • Non-patent document 4 Cellular and Molecular Neurobiology, (USA), 2004, No. 24, No. 4, p.517-533
  • Patent Document 5 Journal of Neurochemistry, (Journal of Neurochemistry), ( (UK), 2002, 81st, No. 3, p. 565-574
  • Non-Patent Document 6 European Journal of Neuroscience, (France), 2004, No. 20, No. 11, p. 3035-3040
  • Non-Patent Document 7 Journal Obob 'Chromatography B: Biomedical' Sciences
  • Non-Patent Document 8 Endocrinology, (USA), 1994, No. 135, No. 6, p. 2742-2748
  • An object of the present invention is to provide a therapeutic agent for eating disorders or drinking disorders containing, as an active ingredient, a peptide having an activity that promotes eating or drinking behavior, and using the peptide, It is to provide a screening method for substances that inhibit or promote the activity.
  • the present invention relates to the following (1) to (6).
  • a therapeutic agent for eating disorders or drinking disorders comprising at least one peptide selected from the following (a) to (e) or a pharmacologically acceptable salt thereof.
  • R 1 represents a hydrogen atom, a substituted or unsubstituted alkanol, a substituted or unsubstituted aroyl, a substituted or unsubstituted heteroarylcarbonyl, a substituted or unsubstituted alkoxycarbonyl, substituted or Represents unsubstituted aryloxycarbonyl, or substituted or unsubstituted heteroaryloxycarbonyl
  • R 2 represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino
  • A represents (A) to (d) above, which represents a peptide residue of one of the peptides).
  • the peptide or salt thereof described in any one of the above and a test substance are brought into contact with hypothalamic cells, pituitary cells or brain tissue.
  • the cellular response is suppressed compared to the cellular response when the peptide or its salt is contacted with hypothalamic cells, pituitary cells or brain tissue in the absence of the test substance.
  • the test substance is selected as a substance that inhibits the activity of promoting the feeding or drinking behavior of the peptide, and the activity of promoting the feeding or drinking behavior of the peptide Screening method for substances that inhibit the above.
  • the peptide described in any one of the above or a salt thereof and a test substance are brought into contact with hypothalamic cells, pituitary cells or brain tissue.
  • the cellular response is measured and the cellular response is enhanced compared to the cellular response when the peptide or salt thereof is contacted with hypothalamic cells, pituitary cells or brain tissue in the absence of the test substance
  • the test substance is selected as a substance that promotes the activity of promoting the feeding or drinking behavior of the peptide, which promotes the activity of promoting the feeding or drinking behavior of the peptide Screening method for substances to be used.
  • the peptide or salt thereof described in any one of the above and the test substance may be hypothalamic cells, pituitary cells, or hypothalamic cells.
  • the amount of the peptide or its salt bound to the cell or the membrane fraction of the cell when contacted with the membrane fraction of pituitary cells is measured, and the peptide or its salt is measured in the absence of the test substance.
  • the test substance when the binding amount is lower than the binding amount of the peptide or a salt thereof when contacted with a cell or a membrane fraction of the cell as an agonist or antagonist for the receptor of the peptide A method for screening an agonist or antagonist for a receptor of the peptide, which comprises selecting.
  • a therapeutic agent for eating disorders or drinking disorders containing a peptide having an activity of promoting eating or drinking behavior as an active ingredient, and the use of the peptides using the peptides.
  • Methods of screening for agonists or antagonists for substances that promote or suppress activity or receptors for the peptides are provided.
  • FIG. 1 shows an increase in intracellular calcium concentration in hypothalamic cells of apoaequorin-expressing mice by 1 ⁇ mol / L of peptide 1 (peptide represented by SEQ ID NO: 1).
  • the horizontal axis is the time after addition of the medium (seconds), and the vertical axis is the relative light output (RLU) per second.
  • Peptide 1 was added in 25 seconds.
  • FIG. 2 shows the increase of intracellular calcium concentration in pituitary cells of apoaequorin-expressing mice by 5 / i mol / L of peptide 1.
  • the horizontal axis is the time after addition of the medium (seconds), and the vertical axis is the relative light output (RLU) per second.
  • Peptide 1 was added at 25 seconds.
  • FIG. 3 shows an increase in intracellular calcium concentration in hypothalamic cells of apoaequorin-expressing mice by 1 / i mol / L of peptide 2 (peptide represented by SEQ ID NO: 2).
  • the horizontal axis is the time after addition of the medium (seconds), and the vertical axis is the relative light output (RLU) per second. In 25 seconds Peptide 2 was added.
  • FIG. 4 shows the increase in intracellular calcium concentration in pituitary cells of apoaequorin-expressing mice by 1 / i mol / L of peptide 2.
  • the horizontal axis is the time after addition of the medium (seconds), and the vertical axis is the relative light output (RLU) per second.
  • Peptide 2 was added in 25 seconds.
  • FIG. 5 shows promotion of feeding behavior of rats by peptide 1. The results are one hour after peptide administration. The vertical axis is food intake (g).
  • FIG. 6 shows promotion of water intake behavior of rats by peptide 1. The results are one hour after peptide administration. The vertical axis is the water intake (g).
  • FIG. 7 shows promotion of rat water intake behavior by peptide 2 and peptide 3 (peptide represented by SEQ ID NO: 5).
  • the vertical axis is the water intake (g).
  • the results for the mouth are 1 hour after the administration of the peptide, and the results for the country are 2 hours after the administration of the peptide.
  • the therapeutic agent for eating disorders or drinking disorders of the present invention contains any of the following peptides (a) to (e) or a pharmacologically acceptable salt thereof as an active ingredient.
  • the following peptides (a) to (e) are also referred to as “VGF-related peptides”.
  • a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 6.
  • the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence 1J of human amino acid 586 to 615 of human VGF
  • the amino acid sequence of SEQ ID NO: 2 is a sequence corresponding to the amino acid sequence of SEQ ID NO: 1 in the amino acid sequence of rat VGF, Corresponds to amino acid sequence 588-617 of rat VGF.
  • the amino acid sequence of SEQ ID NO: 3 is a consensus sequence of SEQ ID NOS: 1 and 2, and is an amino acid sequence represented by the following formula (II).
  • X 1 and X 2 independently represent any amino acid that is the same or different.
  • X 1 is preferably aspartic acid, glutamic acid, isasparic acid, isoglutamic acid, 2-aminoadipine. Acid or 2-aminosuberic acid
  • X 2 is Noreginine, lysine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid or histidine, more preferably, X 1 is ispartic acid or glutamic acid, and X 2 is arginine or histidine. It is.
  • amino acid sequence of SEQ ID NO: 4 is the amino acid sequence lj of human VGF, positions 554 to 615, SEQ ID NO:
  • the amino acid sequence of 5 corresponds to the amino acid sequence of positions 556-617 of rat VGF.
  • the amino acid sequence of SEQ ID NO: 6 is a consensus sequence of SEQ ID NOs: 4 and 5, and is an amino acid sequence represented by the following formula (III).
  • X 1 is a Asuparagin acid, Gunoretamin acid, Isoasuparagin acid, isoglutamine acid, 2-aminoadipic acid or 2 Aminosu base phosphoric acid
  • X 2 is ⁇ Noreginin, lysine, Ol two Chin 2, 4-diaminobutanoic acid, 2,3-diaminopropionic acid or histidine
  • X 3 , X 4 , X 7 are serine or alanine
  • X 5 is serine or leucine
  • X 6 is Tyrosine or phenylalanine
  • X 8 is tyrosine or histidine
  • X 9 is glycine or aspartate
  • x 1C) is arginine or leucine, more preferably X 1 is ispartate or glutamate.
  • X 2 is arginine or histidine.
  • (b) A peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 6 and having an action of promoting feeding or drinking behavior.
  • the amino acid sequence of the peptide of (b) may contain any number of amino acids as long as it contains any of the amino acid sequences of SEQ ID NOs:! To 6 and has an effect of promoting feeding or drinking behavior. The following is preferred 60 or less is more preferred and 40 or less is particularly preferred.
  • a peptide comprising an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 1, 2, 4, or 5 and having an action of promoting feeding or drinking behavior.
  • R 1 represents a hydrogen atom, a substituted or unsubstituted alkanol, a substituted or unsubstituted aroyl, a substituted or unsubstituted heteroarylcarbonyl, a substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted Represents unsubstituted aryloxycarbonyl, or substituted or unsubstituted heteroaryloxycarbonyl, R 2 represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, A represents Represents a peptide residue of any of the peptides (a) to (d) above.
  • amino acid sequence of SEQ ID NO: 1, 2, 4 or 5 amino acid residues are substituted, deleted or added in any one or more amino acid sequences in the same sequence. This means that there are substitutions, deletions or additions of 1 to 5 amino acid residues, and substitutions, deletions or additions may occur simultaneously.
  • amino acids to be substituted or added include, for example, 20 L-amino acids known as essential amino acids, that is, L-alanine, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L -Glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenenolealanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, Examples include, but are not limited to, L-parin and L-cystine, such as tert-leucine, noroleucine, norpaline, 2-aminobutanoic acid, ⁇ methylserine, t_butylglycine, t-butylalanine, cyclohexane Xylalanin, isoaspartic acid, iso
  • Group A Leucine, isoleucine, norleucine, valine, norpaline, alanine, 2-aminobutanoic acid, methionine, monomethylserine, t_butylglycine, t-butylalanine, cyclohexylalanine, tert-leucine
  • Group B aspartic acid, gnoretamic acid, isospartic acid, isoglutamic acid, 2-amino adipic acid, 2_aminosuberic acid
  • Group D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E Proline, 3-hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • Group G phenylalanin, tyrosine
  • amino acid sequence IJ having a homology of 90% or more with the amino acid sequence of SEQ ID NO: 1, 2, 4 or 5 refers to the homology analysis program BLAST 2 Sequences [FEMSMicrobiol Lett. 174, 247 (1999)].
  • alkanoyl includes, for example, linear or branched carbon atoms having 1 to 20, for example, formyl, acetyl, propionyl, butyryl, isobutyryl. , Norelinore, isovaleryl, pivalol, hexanol, heptanol, lauroinor, eicosanoyl and the like.
  • aryl moiety of the aroyl and allyloxycarbonyl examples include, for example, vinyl and naphthyl having 6 to 15 carbon atoms.
  • Heteroarylcarbonyl and heteroaryloxycarbonyl heteroary For example, furyl, chenyl, pyrrolyl, pyrazolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinole, pyridazinyl, pyrimigenole, pyrazol, indolyl, indazolyl, benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxylinyl, Can be given.
  • alkoxycarbonyl and the alkyl moiety of alkoxy include, for example, linear or branched carbon atoms having 1 to 20 carbon atoms such as methyl, ethyl, propyl, isopropyl, butanol, pentyl, hexyl, heptyl, decyl, Examples include dodecyl and eicosyl.
  • Substituents in the substituted alkanoyl, substituted alkoxycarbonyl and substituted alkoxy may be the same or different and the number of substitutions:!
  • cycloaliphatic alkyl such as cycloheptyl and cyclooctyl, substituted or unsubstituted phenol, fluorenyl and the like.
  • substituent of the substituted phenyl include alkyl, alkoxy, hydroxy, nitro, sulfo, cyan, halogen, etc., which are the same or different and have 1 to 3 substitutions.
  • the halogen include fluorine, chlorine, bromine and iodine. Each atom is raised.
  • the alkyl part of the substituted phenyl and the alkyl part of alkoxy are the same as the alkyl part of alkoxycarbonyl and alkoxy.
  • Substituents in substituted aroyl, substituted aryloxycarbonyl, substituted heteroarylcarbonyl, and substituted heteroaryloxycarbonyl are the same or different and have 1 to 3 substituents. It is synonymous with the substituent of.
  • substituent in the substituted amino examples include the same or different substituents having 1 to 2 substituents such as substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and the like.
  • the alkynole has the same meaning as the alkyl moiety such as alkoxy
  • the substituent of the substituted alkyl has the same meaning as the substituent such as the substituted alkoxy.
  • Aaryl is synonymous with the aryl moiety of the above-described aryl or aryloxycarbonyl
  • the substituent of the substituted aryl is synonymous with the substituent of said aryl or aryloxycarbonyl.
  • amino acid residues whose side chain functional group is chemically modified or protected include, for example, a side chain carboxy group protected with a benzyl ester.
  • amino acid residues whose side chain functional group is chemically modified or protected include, for example, a side chain carboxy group protected with a benzyl ester.
  • examples include aspartic acid residues or gnoretamic acid residues, and cysteine residues in which the side chain thiol group is carboxymethylated.
  • Examples of pharmacologically acceptable salts include acid addition salts, metal salts, organic base addition salts, and the like.
  • Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate and phosphate, and organic acid salts such as acetate, maleate, fumarate, tartrate and kenate.
  • Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like.
  • organic base addition salt examples include primary amines such as methenoreamine, ethylamine, and aniline, secondary amines such as dimethylamine, jetylamine, pyrrolidine, piperidine, morpholine, and piperazine, trimethylenoleamine, and triethinorea. And salts formed with tertiary amines such as amine, N, N-dimethylaniline and pyridine, and ammonium salts.
  • primary amines such as methenoreamine, ethylamine, and aniline
  • secondary amines such as dimethylamine, jetylamine, pyrrolidine, piperidine, morpholine, and piperazine, trimethylenoleamine, and triethinorea.
  • salts formed with tertiary amines such as amine, N, N-dimethylaniline and pyridine, and ammonium salts.
  • the fact that the peptides (a) to (e) have the activity of promoting feeding behavior or water feeding behavior includes that the peptide is contained in the lateral ventricle of the rat shown in Example 2.
  • Administer physiological saline measure the amount of food and water consumed for 1 to 2 hours after administration, and compare with the amount of food and water consumed when only physiological saline is administered. This can be confirmed by an increase in food intake or water consumption when physiological saline containing peptides is administered compared to when physiological saline alone is administered. It is also possible to confirm that the peptides (a) to (e) have the activity of increasing the intracellular calcium ion concentration in hypothalamic cells or pituitary cells.
  • the peptide has an activity to increase the intracellular calcium ion concentration of hypothalamic cells or pituitary cells indicates that the hypothalamus or pituitary gland extracted from a transgenic mouse expressing apoaequorin as shown in Example 1.
  • Shred and culture in a medium containing coelenterazine add the medium, add the medium containing the peptide 25 seconds later, and measure the relative luminescence with a luminometer immediately after the addition of the medium. This can be confirmed by an increase in relative light emission when a medium containing a peptide is added compared to when a medium is added.
  • VGF-related peptides include, for example, Nobuo Izumiya, Tetsuo Kato et al., “Basics and Experiments of Peptide Synthesis”, Maruzen, (1985), Saburo Aimoto et al., “Experimental Chemistry Course”, 4th edition, No. 22 ⁇ , “Yes Machine Synthesis IV Acid 'Amino Acid' Peptide ”, Maruzen, (1999), Int. J. Pept. Protein Res. 35, 1 61-214 (1990), Fields, GB, Solid-Phase Peptide Synthesis, Methods in Enzymolog y , vol.
  • the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a dichloromethane method, an active ester method, a carboimidazole method, and a redox method.
  • the synthesis can be achieved by applying both solid phase synthesis and liquid phase synthesis methods. That is, when the amino acid constituting the peptide of the present invention and the remaining part are condensed and the product has a protecting group, the desired peptide can be synthesized by removing the protecting group.
  • VGF-related peptide is one in which the side chain of the amino acid residue constituting the peptide and Z or the amino terminal of the peptide and / or the carboxy terminal of the peptide are chemically modified or protected.
  • side chain of the amino acid residue constituting the peptide and Z or the amino terminal of the peptide and / or the carboxy terminal of the peptide are chemically modified or protected.
  • a method of chemically modifying after peptide synthesis such as a method of chemically modifying after peptide synthesis, a method of peptide synthesis using chemically modified amino acids, or a method of appropriately selecting the reaction conditions for final deprotection of peptide synthesis.
  • VGF-related peptide can also be synthesized using an automatic peptide synthesizer.
  • Peptide synthesis using a peptide synthesizer is carried out using a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation or a peptide synthesizer manufactured by Advanced ChemTech, Inc.
  • Fmoc (9-fluorenylmethyloxycarbonyl) monoamino acid or N-Boc (t_butyloxycarbonyl) monoamino acid can be used according to the respective synthesis programs.
  • Mino acids and carrier resins are applied in Applied Biosystems, Shimadzu, Kokusan Chemicals, EMD Biosienses, Inc., Watanabe Chemical, Advanced Chemtech, Anaspec (AnaSpec, In) or Peptide Research Institute Co., Ltd.
  • VGF-related peptide can be performed by a combination of conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.
  • a pharmaceutical preparation containing a VGF-related peptide or a pharmacologically acceptable salt thereof is an active ingredient of the peptide or a pharmacologically acceptable salt thereof alone or as an active ingredient for any other treatment. And a mixture thereof.
  • These pharmaceutical preparations are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers.
  • the administration route can be oral or the parenteral, for example, intracerebroventricular or intravenous, as it is desirable to use the most effective treatment.
  • Administration forms include tablets, powders, granules, syrups, injections and the like.
  • Liquid preparations suitable for oral administration such as syrups, include sugars such as water, sucrose, sonorebit, fructose, dalicols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil and soybean oil. And preservatives such as p-hydroxybenzoates, and flavors such as strawberry flavor and peppermint.
  • Tablets, powders and granules include excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as a fatty acid ester, and a plasticizer such as glycerin.
  • Formulations suitable for parenteral administration preferably comprise a sterile aqueous solution containing the active compound that is isotonic with the blood of the recipient.
  • a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
  • a carrier comprising a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
  • these parenteral agents one kind selected from diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., exemplified for oral agents. Or more auxiliary components can be added.
  • the dosage and frequency of administration of the VGF-related peptide or pharmacologically acceptable salt thereof varies depending on the dosage form, patient age, body weight, nature or severity of the symptom to be treated.
  • 0. Olmg to: lg preferably 0.05 to 50 mg per adult is administered once to several times a day.
  • parenteral administration such as intravenous administration, per adult, 0.001 to:! OOmg, preferably f or 0.01 to:! Omg is administered several times at a time.
  • the dose and the number of doses vary depending on the various conditions described above.
  • the VGF-related peptide has an activity of promoting feeding or drinking behavior
  • the above pharmaceutical preparation is used for eating disorders such as anorexia nervosa (anorexia nervosa, anorexia nervosa) or drinking water.
  • eating disorders such as anorexia nervosa (anorexia nervosa, anorexia nervosa) or drinking water.
  • VGF-related peptides have the activity of promoting feeding or drinking behavior, the peptide acts on the brain, particularly the hypothalamus and pituitary gland, which are organs involved in the control of feeding or drinking behavior. , It is thought to cause the cellular response.
  • the substance that inhibits the activity of the VGF-related peptide to promote feeding or drinking behavior is as follows: (i) contact the hypothalamic cell, pituitary cell or brain tissue with the VGF-related peptide or salt thereof and the test substance. (Ii) compared with the cellular response when the peptide or its salt is contacted with the same cell in the absence of the test substance, and (iii)
  • test substance When the cellular response is suppressed in the presence of a test substance, the test substance can be screened by selecting it as a substance that inhibits the activity of promoting the feeding or drinking behavior of the peptide.
  • a cellular response when a VGF-related peptide or a salt thereof and a test substance are contacted with hypothalamic cells, pituitary cells or brain tissue is measured, and (ii) the same cells are tested.
  • the test substance is compared with the cellular response when the peptide or its salt is contacted in the absence of the substance.
  • the cellular response may be any measurable cellular response that occurs when a VGF-related peptide is brought into contact with hypothalamic cells, pituitary cells, or brain tissue. Increase in intracellular calcium ion concentration.
  • the hypothalamic cells and pituitary cells used in the above screening method may be cell lines derived from the hypothalamus or pituitary as long as they show a cellular response when contacted with a VGF-related peptide,
  • the hypothalamus or pituitary gland from animals may be shredded. Further, a part of the brain including the hypothalamus or the pituitary gland may be shredded.
  • the brain tissue used in the above screening method is preferably a part of the brain including the hypothalamus or the pituitary gland, or a part of the whole brain that has been shredded.
  • hypothalamus or pituitary gland of a transgenic mouse (WO02 / 010371) that expresses apoaequorin produced in the whole body by introducing the apoaequorin gene, and the cells obtained by shredding are luminescent in the presence of coelenterazine. It is preferable to measure the intracellular calcium ion concentration by measuring with a luminometer.
  • the VGF-related peptide obtained by the above screening method has an activity that promotes feeding or drinking behavior, as well as the VGF-related peptide. So it can be used as a therapeutic agent for diseases such as eating disorders and drinking disorders.
  • a substance that inhibits the activity of promoting the feeding or drinking behavior of the VGF-related peptide obtained by the above screening method suppresses the feeding behavior. Therefore, it is useful for treating obesity or treating obesity. It can be used as a drug to support accompanying diet.
  • An agonist or antagonist for a receptor for a VGF-related peptide is: (i) the cell when the VGF-related peptide or a salt thereof and a test substance are contacted with a hypothalamic cell or pituitary cell or a membrane fraction of the cell or For the membrane fraction of the cell. The amount of binding of the peptide or its salt, and (ii) the same cell or membrane fraction against the cell or membrane fraction of the cell when the peptide or salt thereof is contacted in the absence of the test substance.
  • test substance when the binding amount of the peptide or salt thereof decreases in the presence of the test substance, the test substance is selected as an agonist or antagonist for the receptor of the peptide Can be screened.
  • screening can be performed by a similar method using brain tissue or membrane fraction of brain tissue instead of hypothalamic cells or pituitary cells or membrane fractions of the cells.
  • the hypothalamic cell, pituitary cell, and brain tissue used in the above screening the cell and brain tissue described in 3. above can be used.
  • the cells or cell membrane fraction of the cells, or brain tissue or membrane fraction of brain tissue are suspended in a suitable buffer.
  • the buffer may be any buffer that does not inhibit the binding of the VGF-related peptide or salt thereof to the suspended cell, tissue or membrane fraction, eg pH 4 to 10 (preferably pH 6 to 8) Phosphate buffer or Tris-HC1 buffer.
  • various proteins such as CHAPS, Tween-80, digitonin, deoxycholic acid, and other surface active agents such as urushi serum albumin and gelatin can be added to the buffer.
  • a protease inhibitor such as PMSF, leupeptin, E-64, or pepstatin may be added for the purpose of suppressing degradation of the polypeptide or ligand of the present invention by protease.
  • the binding amount of the VGF-related peptide or a salt thereof to the cell or membrane fraction is, for example, by labeling the peptide and bringing it into contact with the cell or the membrane fraction of the cell. It can be measured as the amount of label bound to the membrane fraction.
  • the labeling substance include a radioisotope, a fluorescent substance, a luminescent substance, piotin, digoxigenin, a peptide containing a tag sequence, an enzyme, and the like, and the labeled peptide is the cell or a membrane fraction of the cell. Anything, as long as it has a bond. Specific examples of screening methods are given below.
  • the radioactivity at this time is defined as the total binding amount ( ⁇ ).
  • the same reaction is carried out under the condition where a large excess of the unlabeled same peptide or its salt is added, and the radioactivity at that time is defined as the nonspecific binding amount ( ⁇ ).
  • ⁇ — ⁇ is the amount of binding in the absence of the test substance. Perform the same measurement as the total amount of binding under the condition that the test substance is added, and let C be the radioactivity at that time.
  • C—B is the amount of binding in the presence of the test substance.
  • the binding inhibition rate of the test substance can be obtained by the following formula.
  • Inhibition rate (%) [1 _ ⁇ (C_B) / (A-B) ⁇ ] X 100
  • the agonist for the receptor of the VGF-related peptide obtained by the above screening method has an activity that promotes eating or drinking behavior. It can be used as a therapeutic agent for diseases.
  • the antagonist to the receptor inhibits the activity of the VGF-related peptide to promote eating behavior, so that it supports a therapeutic agent for obesity or diet associated with the treatment of obesity. It can be used as a drug.
  • Peptides consisting of the amino acid sequences of SEQ ID NOS: 1, 2 and 5 (hereinafter referred to as Peptide 1, Peptide 2 and Peptide 3, respectively) were chemically synthesized by requesting from Sigmaji Anosys.
  • the amino acid sequence of SEQ ID NO: 1 is amino acid sequence 586 to 615 of the amino acid sequence of human VGF
  • the amino acid sequence of SEQ ID NO: 2 is amino acid sequence 588 to 617 of the amino acid sequence of rat VGF
  • the amino acid sequence of 5 is the 556th to 617th sequence of the amino acid sequence of rat VGF.
  • apoaequorin-expressing mouse By using hypothalamic cells and pituitary cells of a transgenic mouse (hereinafter referred to as an apoaequorin-expressing mouse) of the apoaequorin gene that expresses apoaequorin throughout the body, the effect of the peptide on these cells is demonstrated.
  • the intracellular calcium ion concentration was used as an index.
  • Apoaequorin-expressing mouse cells show that In the presence of interterazine, it binds to calcium ions and emits light, so the intracellular calcium ion concentration can be monitored.
  • the patent document (WO02 / 010371) describes a method for producing an apoetaline-expressing mouse, a method for evaluating a physiologically active substance using a biological sample derived from the mouse, and a physiologically active peptide as shown below in the organ of the mouse. The experimental results evaluated using are disclosed.
  • Apoaequorin-expressing mice were prepared according to the method disclosed in Reference Example 4 of the patent document (WO02 / 010371). After apoaequorin-expressing mice were sacrificed, the hypothalamus and pituitary gland were excised, and each sample was prepared by cutting into approximately 1 to 2 mm 3 cubes. Next, in each hypothalamic sample or pituitary sample, RPMI1640 medium was used in a 5 mL tube (Rohren-Tubes; manufactured by Sarstedt, No. 55. 476). 50 mol of mol / L coelenterazine [Molecular Probes (MolecularProbes)] solution and 3 prepared samples were added and incubated at 37 ° C for 3 hours.
  • RPMI1640 medium was added, and 25 seconds later, the peptide dissolved in RPMI1640 medium was terminated for the pituitary sample so that the final concentration was 1 ⁇ mol / L for the hypothalamic sample.
  • peptides 1 and 2 have the activity of increasing intracellular calcium ion concentration in hypothalamic cells and pituitary cells. Since the hypothalamus and pituitary gland are organs involved in the appetite center that govern feeding and drinking behavior, peptides 1 and 2 act on hypothalamic cells and pituitary cells to cause feeding or feeding. Expected to affect water behavior It was.
  • physiological saline or artificial cerebrospinal fluid 10 ⁇ 1 was administered into the lateral brain cortex under the same conditions as the peptide administration by changing the day to the same rat, and under free action for 1 hour or 2 hours after administration. Water consumption and food intake were measured. The amount of water consumed and the amount of food consumed at the time of peptide administration were compared with the amount of water consumed and the amount of food consumed at the time of administration of the control physiological saline by means of their respective average values.
  • a therapeutic agent for eating disorders or drinking disorders containing a peptide having an activity of promoting feeding or drinking behavior as an active ingredient, and the use of the peptides using the peptides
  • Methods of screening for agonists or antagonists for substances that promote or suppress activity or receptors for the peptides are provided.
  • the substance that promotes the activity of the peptide obtained by the screening method provided by the present invention and the agonist for the receptor of the peptide are useful for the treatment of eating disorders or drinking disorders, and the activity of the peptide Inhibiting the substance and antagonists to the receptor of the peptide Is useful for the treatment of obesity, or diet therapy associated with obesity treatment.
  • SEQ ID NO: 1 Inventor: Masamitsu Nakazato; Motoo Yamazaki
  • SEQ ID NO: 3 Consensus sequence of amino acid sequences SEQ ID NO: 1 and 2
  • SEQ ID NO: 6 Consensus sequence of amino acid sequences SEQ ID NO: 4 and 5

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Abstract

La présente invention concerne un agent thérapeutique pour un trouble de PRISE alimentaire ou un trouble de consommation d'eau, qui comprend en tant que principe actif un peptide dont l'activité consiste à stimuler le comportement de prise alimentaire ou le comportement de consommation d'eau. La présente invention concerne également un procédé de criblage d'une substance capable de renforcer ou d'inhiber l'activité dudit peptide, basé sur l'utilisation de ce peptide.
PCT/JP2007/053267 2006-02-22 2007-02-22 Agent therapeutique pour un trouble de prise alimentaire ou un trouble de consommation d'eau WO2007105442A1 (fr)

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JP2008505024A JPWO2007105442A1 (ja) 2006-02-22 2007-02-22 摂食障害または摂水障害の治療薬

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1939286A1 (fr) * 2005-07-29 2008-07-02 Kyowa Hakko Kogyo Co., Ltd. Nouveaux peptides

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WO2001064835A2 (fr) * 2000-02-28 2001-09-07 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
JP2003505471A (ja) * 1999-07-21 2003-02-12 アムジエン・インコーポレーテツド Ｖｇｆポリペプチドおよびｖｇｆ関連障害の治療方法
WO2005047484A2 (fr) * 2003-11-07 2005-05-26 Ciphergen Biosystems, Inc. Biomarqueurs pour la maladie d'alzheimer biomarkers for alzheimer's disease
WO2007013586A1 (fr) * 2005-07-29 2007-02-01 Kyowa Hakko Kogyo Co., Ltd. Nouveau peptide

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
JP2003505471A (ja) * 1999-07-21 2003-02-12 アムジエン・インコーポレーテツド Ｖｇｆポリペプチドおよびｖｇｆ関連障害の治療方法
WO2001064835A2 (fr) * 2000-02-28 2001-09-07 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2005047484A2 (fr) * 2003-11-07 2005-05-26 Ciphergen Biosystems, Inc. Biomarqueurs pour la maladie d'alzheimer biomarkers for alzheimer's disease
WO2007013586A1 (fr) * 2005-07-29 2007-02-01 Kyowa Hakko Kogyo Co., Ltd. Nouveau peptide

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Title
HAHM S. ET AL.: "VGF is required for obesity induced by diet, gold thioglucose treatment, and agouti and is differentially regulated in pro-opiomelanocortin- and neuropeptide Y-containing arcurate neurons in response to fasting", J. NEUROSCI., vol. 22, no. 16, 2002, pages 6929 - 6938, XP003017738 *
LIU J.-W. ET AL.: "Peptide V: a VGF-derived neuropeptide purified from bovine posterior pituitary", ENDOCRINOLOGY, vol. 135, no. 6, 1994, pages 2742 - 2748, XP000953047 *
TRANI E. ET AL.: "Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor", J. NEUROCHEM., vol. 81, no. 3, 2002, pages 565 - 574, XP003006590 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1939286A1 (fr) * 2005-07-29 2008-07-02 Kyowa Hakko Kogyo Co., Ltd. Nouveaux peptides
EP1939286A4 (fr) * 2005-07-29 2009-05-20 Kyowa Hakko Kirin Co Ltd Nouveaux peptides

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